BMS III:

Cellular Organization and Function

Chapter 11: Transmembrane Transport of Ions

and Small Molecules

Lecture 1: Membrane Transport

Membrane Potential
Ion Channels
Total 13 marks:
5 mark will be online exam: 20 MC
8 mark will be mid term exam:
1
Essay type question (3)
4
short question (4)
4 MC
(1)

CHAPTER 11
Transmembrane Transport of Ions and Small Molecules

© 2021 W. H. Freeman and Company

 Cell membrane:
• Cell membrane regulate the cellular structure and
function.
• In all organisms, cellular membranes are composed of
phospholipid bilayer, with hydrophilic (water loving)
head and a hydrophobic ( water opposing) tail.
• Cell membrane contain different proteins including
membrane transport proteins for enabling specific
ion, sugar, to enter and exit the cell.
• Small molecules including water and ions serve as
substrate for many reactions which takes place inside
cell (energy metabolism, cell signaling).
Membranes
For life to be sustained, cells must rigorously
maintain
their volume and internal composition.
Proper
functioning of
Glucose any cell
depends on
ATP precise control
of import and
export of
various
NH3 molecules and
H2O ions.
H2O

This is accomplished by the cell membrane and its transport

properties.
 Significance
Physical and clinical significance:
• Forms permeability barrier that separates intracellular cytoplasm and
other organelle from extracellular environment for regulating movement
of molecules and ions.
• Made of phospholipid bilayer in which other types of specific lipid and
protein molecules are embedded.?
• Maintaining membrane potential (proton gradient, chemical gradient,
acidic environment).
• Gas exchange (lung)
• Action potential in neuronal cells
• Cell signaling.
• In flux and efflux of drugs (Multi drug resistance).
• Clinical relevance: Cystic fibrosis, gastric ulcers, channel blocker etc.
• Muscle contraction (ECG, BP)
Transport mechanisms:
Question:
• How cells are organized? How they function and their
control mechanism at organ and whole body level?

• What is role of cell membrane in cellular function?

Cell membranes are dynamic and maintain their
property during budding and fusion (cell division,
fusion with lysosome etc.).

Fluid nature of cell membrane allows cells to acquire

different shape for different function (allows RBC to
squeeze through blood vessels, cell migration)
Functions of Membrane:
• Define the boundaries of the cell
• Allow import and export
-Selective import of nutrients (e.g. lactose)
-Selective export of waste and toxins (e.g. antibiotics)
• Retain metabolites and ions within the cell
• Sense external signals and transmit information into the cell
• Provide compartmentalization within the cell
1. separates energy-producing reactions from energy- consuming
2. keeps proteolytic enzymes away from cellular proteins
• Produce and transmit nerve signals
• Store energy as a proton gradient and support synthesis of ATP
 Implication in physiology:
• Increased tumor growth
• Antibiotic resistance
• Muscle contraction
• Nerve conduction
• Heart burn/channel blocker
• Ulceration
• Urine secretion
• Insulin secretion
• Cholera
Water follows the salt and sugar: Failed regulation cause many disease
(hypertension, diabetes, diarrhea, fluid buildup in liver & kidney failure).
Think about Sodium/Potassium levels significance during blood test.
Think about use of saline/buffered salt solution while working with cells.
Acidic environment (pH change/proton gradient)
• Homologous expressed channel proteins can be activated by
light (optogenetic) or other drugs (chemogenetic) for disease
treatment.
• Epilepsy caused by mutation in ion channel gene, called
channelopathies.
• Topical anaesthetic Lidocaine works by blocking flow of Na+.
• Ion channels are responsible for odor, taste, nerve induction
etc.
 Figure 23.2, Page 1045

Representation of a normal EKG on the

left with normal intervals labeled. The P-
wave represents atrial activity, the QRS
represents ventricular activation, and the
QT interval represents ventricular
recovery, or repolarization. Note the
panel on the right, typical of a patient
with Long-QT syndrome. The QT interval
depicted here is significantly prolonged,
with ventricular repolarization delayed,
leading to an increased risk of ventricular
tachyarrhythmias.

https://www.washingtonhra.com/arrhythmias/long-qt-syndrome.php
 Major Functions of Biological Membranes:
• permit shape changes that accompany cell growth and
movement
• permit exocytosis, endocytosis, and cell division
• serve as molecular gatekeepers
Proteins and Enzymes In and On the Membranes:
• transporters = move specific organic solutes and
inorganic ions across the membrane
• receptors = sense extracellular signals and trigger
molecular changes in the cell
• ion channels = mediate electrical signaling between
cells
• adhesion molecules = hold neighboring cells together
 Membrane Trafficking
• membrane trafficking = process by which membrane
lipids and proteins that are synthesized in the ER move
to their destination organelles or to the plasma
membrane

• lipids and proteins undergo covalent modifications in the

Golgi apparatus
– dictates the eventual location of the mature protein
Membranes

Polar

Nonpolar
 Membrane Proteins Differ in the Nature of
Their Association with the Membrane Bilayer
• integral membrane proteins = firmly embedded within
the lipid bilayer

• peripheral membrane proteins = associate with the

membrane through electrostatic interactions and
hydrogen bonding

• amphitropic proteins = associate reversibly with

membranes
– found in both membranes and the cytosol
Integral, Peripheral, and Amphitropic Proteins

• monotopic = interact with only a single leaflet of the membrane (COX2).

• bitopic = span the bilayer once, extending on either surface (Glycophorin in RBC)
• polytopic = cross the membrane several times. (bacteriorhodopsin)
• GPCRs (seven-transmembrane (7tm) or heptahelical receptors) span the membrane
seven times
– interact with heterotrimeric G proteins
• The biological membrane is a lipid bilayer with proteins of
various functions (enzymes, transporters) embedded in or
associated with the bilayer. Membrane proteins are associated
with the lipid bilayer more or less tightly, and proteins and lipids
are both allowed limited lateral motion in the plane of the bilayer.
• Lipid rafts are functionally specialized regions with unique lipid
and protein compositions.
• Although the lipid bilayer is impermeable to charged or polar
solutes, cells of all kinds have many membrane transporters
and ion channels that catalyze transmembrane movement of
specific solutes. Some transporters simply speed the movement
of solutes in the direction that simple diffusion takes them,
whereas others use an energy source to move solutes against a
concentration gradient.
Different cell types are able to perform different function.
• Cell membrane regulate the entry and exit of
metabolites and ions as per specific cellular need.
• Membrane composition is regulated in different cell
types to perform different specialized functions:
ciliated gut epithelium, blood cells, neurons, muscle
etc.
Transport mechanism:
• Passive transport (no energy expenditure)
Simple diffusion
Facilitated diffusion
Osmosis
• Active transport: Energy is required (pumps)
Membranes

History of Membrane Composition and

Structure
1925 Gorter and Grendel -
Erythrocyte membranes are a
bilayer

1935 Davson and Danielli -

Bilayer has inner and outer protein
layers

1972 Singer and Nicholson –

Fluid mosaic model
 Importance:
The Nobel Prize in Physiology or Medicine 1991 was
awarded jointly to Erwin Neher and Bert Sakmann "for
their discoveries concerning the function of single ion
channels in cells."

Erwin Neher Bert Sakmann

Figure 1. Registration of the flow of current through single ion channels using the recording
technique of Neher and Sakmann. A schematically shows how a glass micropipette is brought
in contact with the cell, and B, using a higher magnification, a part of the cell membrane, with
ion channels, in close contact with the tip of the pipette. The interior of the pipette is
connected to an electronic amplifier. C shows a channel in greater magnification with its
receptor facing the exterior of the cell and its ion filter. D shows the current passing through
the ion channel as it opens.
 Importance:
The Nobel Prize in Chemistry 1997 was divided, one
half jointly to Paul D. Boyer and John E. Walker "for
their elucidation of the enzymatic mechanism
underlying the synthesis of adenosine triphosphate
(ATP)" and the other half to Jens C. Skou "for the
first discovery of an ion-transporting enzyme, Na+,
K+ -ATPase." Many of the cell\'s functions (nerve
impulses, muscular contractions and
digestion) require that the concentration of
potassium ions inside the cell is higher than
outside it, whereas the concentration of
sodium ions must be lower inside than
outside. It takes a great deal of energy to
bring this about. The energy is stored in a
special substance, adenosine triphosphate
(ATP). In 1957 Jens Christian Skou
discovered an enzyme, Na+/K+-ATPase, that
serves as a sort of biological pump to
transport ions.
 Importance:
Two Investigators of Pores in Cell Membranes (Peter
Agre and Roderick MacKinnon) Win Nobel Prize in
Chemistry—2003
Figure 1. Aquaporin-1, in
ribbon representation, looking
down the central channel. The
eight alpha helices that make
up each of the tetramer’s four
subunits are designated H1–
H8.
(Courtesy of Yoshinori
Fujiyoshi.)

Agr Figure 2. The narrow entrance of

e a K⊕ channel has four sites where
negatively charged oxygen atoms
stabilize K⊕ ions (green). The ions’
mutual electrostatic repulsion
pushes them through the entrance
and down into the water-filled
cavity,where water molecules
provide electrostatic
stability. (Courtesy of Roderick
MacKinnon MacKinnon.)
 Overview

11.1 Overview of Transmembrane Transport: (ATP –

Powered pumps, Ion Channels and Transporters)
11.2 Facilitated Transport of Glucose and Water
11.3 ATP-Powered Pumps and the Intracellular Ionic
Environment
11.4 Nongated Ion Channels and the Resting membrane
Potential
11.5 Cotransport by Symporters and Antiporters
11.6 Transcellular Transport
11.1 Overview of Transmembrane Transport:
• Cellular membranes regulate the traffic of molecules
and ions in and out of cells and their organelles
mediated by membrane transport proteins.
• The simple diffusion rate of substance movement across
the phospholipid part of a membrane is proportional to
its concentration gradient and hydrophobicity.
• Most molecules, except O2 and CO2, are moved by
protein channels and some transporters down their
concentrations gradients or by other transporters and
ATP-powered pumps up their concentration gradients.
11.2 Facilitated Transport of Glucose and Water:
• Protein-mediated transport is faster than simple
diffusion, highly specific, and the transport rate is
limited by the number of transporters (Vmax) and
transporter affinity (Km) for the molecule/ion.
• Water moves by osmosis across membranes but some
membranes have water channels (aquaporin) to
increase the rate of water transport.
• Uniport proteins convert between two conformational
states to facilitate diffusion across a membrane.
11.3 ATP-Powered Pumps and the Intracellular Ionic
Environment:
• Four classes of transmembrane proteins couple
energy released by ATP hydrolysis with energy-
requiring transport of substances against their
concentration gradients.
• The combined action of P-class ATP-powered pumps
generates the usual ionic milieu of animal cells.
• ABC superfamily proteins transport a wide array of
substrates, including toxins, drugs, phospholipids,
peptides, and proteins, into or out of the cell.
11.4 Nongated Ion Channels and the Resting
Membrane Potential:
• The animal cell plasma membrane resting potential is
generated by the ATP-powered Na+/K+ pump and
nongated K+ channels.
• The structure and chemical nature of a channel pore
lowers the activation energy for passage of a specific
ion over other ions, which may be even smaller.
• Patch-clamping techniques measure ion movements
through single channels.
11.5 Cotransport by Symporters and Antiporters:
• Two forces constitute an electrochemical gradient
across a membrane – electric potential and ion
concentration gradient.
• Cotransporters use the energy released by ion (H+/
Na+) movement down its electrochemical gradient to
power transport of another molecule or different ion
up its concentration gradient.
11.6 Transcellular Transport:
• Apical and basolateral plasma-membrane regions of
polarized epithelial cells contain different transport
proteins whose activities are coordinated to
accomplish different transport processes:
– Transcellular transport of amino acids and glucose
from the intestinal lumen to the blood
– Stomach acid secretion
– Bone resorption
 Lipids:
Lipids are structurally diverse class of compounds with low solubility in
water and good solubility in nonpolar solvents.
• storage lipids
• membrane lipids
• signaling lipids
His early work with animal fats revolutionized
the manufacture of soap and of candles.

Membrane lipids are 5-10% of most cell, storage

lipid are >80% of adipocytes.
Eugene Chevreul
Lipids are organic molecules that are August 31, 1786 - April 9, 1889
characterized by low solubility in water http://www.cyberlipid.org/
(relatively hydrophobic).
 Classification of Lipids
Two major categories based on the structure and function:

1. Lipids that contain fatty acids (complex lipids)

can be further separated into:
• storage lipids
• membrane lipids
2.Lipids that do not contain fatty acids: cholesterol, vitamins, pigments etc.

No glycerol
Lipid composition is different in different organelles of
the same cell Rat hepatocyte
–Cholesterol predominant in the
plasma membrane, virtually
absent in mitochondria
–Cardiolipin is a major component
of the inner mitochondrial
membrane but not of the plasma
membrane
–Glycolipids are virtually absent
from animal cells.
Membranes
Membranes
Membranes

The bilayer structure of

biomembranes.

Phospholipid spontaneously form

bilayers

Rail road track orientation

3 class of phospholipid:
Phosphoglycerides (glycerol
derivative)
Sphingolipid (sphingosine
derivative)
Sterol (cholesterol)
Membranes
Membrane proteins are embedded in the lipid bilayer
Both these are required for fluidity and rigidity
Membranes
Organelle membrane is required for maintaining proton
concentration in lysosome, regulate acidity and membrane
potential.

Concentration gradient
Channel
Pump
Membranes
A pure phospholipid bilayer acts as a selectively permeable barrier
Only gases and small uncharged molecules cross membranes by simple
diffusion

Partition coefficient
k is a measure of
hydrophobicity.
Higher k value
means faster travel
across lipid bilayer.
Gradients:

Electrochemical gradients.
• Ions also form an electrical gradient across the membrane, created by the
asymmetric distribution of positively and negatively charged ions across
the membrane.
• The electrical gradient can be in the same or opposite direction from the
chemical gradient.
Diffusion through membranes
Diffusion is the net displacement (transport) of matter from
one region to another due to random thermal motion.

Fick’s law
of diffusion

Rate of molecular migration (diffusion) from o to i = kC io

Rate of molecular migration (diffusion) from i to o = kC ii

Rate of net diffusion across barrier = k(Cio - Cii) = k∆Ci

Thus, the net flow of an uncharged molecule across a permeability barrier
due to diffusion is directly proportional to the concentration difference across
Diffusion through membranes
As ∆x approaches 0, ∆Ci /x approaches dCi /dx so that

Ji = Di [dCi /dx]

This is Fick’s first law of diffusion and simply states that the
rate of flow of an uncharged solute due to diffusion is
directly proportional to the rate of change of concentration
with distance in the direction of flow.

The derivative dCi /dx is the concentration gradient and is

the “driving force” for the diffusion of uncharged particles.
Diffusion through membranes

Ji = Di (dCi /dx)

Ji Slope = Di

dCi /dx
Concentration gradient
Fick’s Law is similar to Ohm’s Law (I = gV)
current = conductance x voltage
flow = conductance x pressure
Diffusion through membranes

Partition coefficient (Ki ) is the ratio of a solute (i )

concentration in an aqueous phase and adjacent
oil or lipid phase at equilibrium.

Equilibrium concentration of i in
Kolive
i =oil
Equilibrium concentration of i in
water

Very important: similar as specificity and affinity in enzymatic reaction

Diffusion through membranes
A model for passive diffusion of small
hydrophobic molecules across a pure
phospholipid bilayer

concentration gradient,
hydrophobicity, distance and
size.
Diffusion through membranes

The size of each point reflects its relative molecular radius.

Diffusion through membranes

The relative diffusion rate of any substance across

membrane is proportional to its concentration
gradient, hydrophobicity and size.

Experiment: How membrane permeability or rate of

difusion can be tested?

Using radioactivity or by labelling molecule of

interest, membrane permeability can be determined.
What are experimental approaches to study the transport
proteins:

Purification and reconstitution in artificial membrane.

Overexpression in recombinant cells.
Diffusion through membranes

The hydrophobicity of a substance is partition

coefficient k, which is equilibrium constant for its
partition between oil and water.

Higher k value (more hydrophobic or more lipid

soluble) faster movement across bilayer.

Ex. Diethylurea (k=0.01) is 50 times more

hydrophobic than urea (k=0.0002) and diffuses
through lipid bilayer 50x faster than urea.
Diffusion through membranes

During a laboratory experiment red-labelled glucose,

blue-labelled water and green labelled ethanol is
added to a solution placed over an artificial, pure
phospholipid membrane. Which color will be
observed on the other side of membrane after 10
min?
D
A-Red (glucose)
B-Blue (water)
C-Green (ethanol)
D-B&C
Diffusion through membranes
Ethanol and Glycine are small molecule of
approximately equal molecular weight. However,
membrane is much more permeable to ethanol than
glycine at pH 7.0. Why?

Ethanol is alcohol, glycine is amino acid (zwitterion)

and dissociate in amino group positive & carboxyl
group negative charge at neutral pH. Charged groups
are impermeable to membrane.
Membrane transport
Evidence for additional transport
mechanisms:

1. Most biological membranes are virtually impermeable to

hydrophilic molecules with five or more carbon atoms
(or atomic radii > 4A) and multivalent ions (e.g.
phosphate). Essential nutrients (glucose, amino acids,
etc.) and ions must enter cells by other mechanisms.

2. Concentrations of water soluble solutes and ions inside

cells are maintained at levels that can not be accounted
for by diffusional processes alone.
Membrane transport
Biological membranes are heterogeneous lipid bilayer having other proteins
 Membrane transport
Three main types
Passive diffusion
Facilitated
diffusion
Active transport

Faster

Slower
Membrane transport
Multiple membrane transport proteins function
together
Gated/non gated channels: Figure 23.9, Page
1052
Membrane transport

Imp. slide
Exterior

Cytosol

ATP – Ion
powered channels Transporters
Pumps
107 – 108 102 – 104
100 – 103 ions/sec molecules/
ions/sec sec
Membrane transport

Energy is expended to move Channels permit movement

ions against their gradient. of ions down their gradient.
Energy comes from ATP Channels are gated or non
hydrolysis. gated (reversible).
Membrane transport

Uniporter transport a specific molecule down its concentration gradient.

Cotransporters (symporter and antiporter) move specific molecules

against their concentration gradient by using the energy supplied by one
or more ions moving down their pre-existing electrochemical gradient.
Membrane transport Imp. slide
Membrane transport
Hydrophilic molecules
Protein transporter is needed - Uniporter
No energy is needed
Depends on concentration gradient
Examples: amino acids, nucleosides, sugars
(glucose)

Faster than simple diffusion

Membrane transport
Uniporters:
Faster than simple diffusion
Partition coefficient is not relevant
Limited number of transporters = Vmax
Reversible
Substrate Specific
Membrane transport Imp. slide

Uniporter-catalyzed transport
• Uniporters accelerate a reaction that is already
thermodynamically favored (similar to enzymes)
• This type of transport is termed facilitated transport or
facilitated diffusion
• Three main features distinguish uniport transport (facilitated
diffusion) from passive diffusion
- The rate of facilitated diffusion is much higher than passive
diffusion (Fick’s Law)
- Transport is substrate specific
- Transport occurs via a limited number of uniporters
(transport exhibits saturation kinetics)
Membrane transport

Binding of glucose (1) triggers conformational change (2) and

release into the cytosol (3).
Transporter then undergoes reverse conformational change
(4).
If concentration gradient reversed, the cycle goes in reverse.
Alternating access mechanism
Membrane transport
Experimental
approaches to study the
transport proteins:

Purified transport proteins can be

studied in artificial bilayer
membranes called liposomes.

Gene encoding specific transport

protein can be expressed at high
levels in cell types that does not
express it.

The difference in transport of a

substance by the transfected and
non transfected cell will be due
to expression of transport
protein.
Diffusion vs Facilitated transport
Transport rate (Km):
Membrane transport
Membrane transport
What will happen if basal glucose concentration is raised to 10mM instead of 5mM.

1.5mM

Km 20.0 mM
GLUT family members

Major
Km Major site of
Protein isoform Proposed function
(mM) expression
(aa)

Ubiquitous Basal glucose uptake:

GLUT1 492 3-7 distribution in tissues transport across blood
and cultured cells tissue barriers

Liver, islets, kidney, High-capacity, low-

GLUT2 524 17
small intestine affinity transport

GLUT3 496 1.4 Brain and nerve cells Neuronal transport

Insulin-regulated
GLUT4 509 6.6 Muscle, adipose, heart transport in muscle
and adipose
Intestine, kidney and
GLUT5 501 Transport of fructose
testis
 transporter
The glucose

family members
Membrane transport

IUBMB Life
Volume 62, Issue 5, pages 315-333, 5 MAR 2010 DOI: 10.1002/iub.315
http://onlinelibrary.wiley.com/doi/10.1002/iub.315/full#fig1
Membrane transport

Physiological role of
hexose transport in
different tissues
Membrane Potential

How membrane potential is generated?

Nernst equation

Patch clamp technique

 Discovery of patch clamp technique:
The Nobel Prize in Physiology or Medicine 1991 was
awarded jointly to Erwin Neher and Bert Sakmann "for
their discoveries concerning the function of single ion
channels in cells."

Erwin Neher Bert Sakmann

Figure 1. Registration of the flow of current through single ion channels using the recording
technique of Neher and Sakmann. A schematically shows how a glass micropipette is brought
in contact with the cell, and B, using a higher magnification, a part of the cell membrane, with
ion channels, in close contact with the tip of the pipette. The interior of the pipette is
connected to an electronic amplifier. C shows a channel in greater magnification with its
receptor facing the exterior of the cell and its ion filter. D shows the current passing through
the ion channel as it opens.
Membrane Potential
Membrane Potential

1M urea 0 urea

1M sucrose 0 sucrose

urea Ju = Du∆Cu/∆x

sucrose Js = Ds∆Cs/∆x

Du > > D s
Membrane Potential

1M potassium 0 potassium
acetate acetate

1M K+
1M Ac-

K+

KAc DK+ > DAc-

Ac- But

DK+ > DKAc > DAc-

Membrane Potential

Law of Electroneurality says that any

macroscopic or bulk portion of a solution must
contain an equal number of opposite changer;
that is, it must be electrically neutral.
Membrane Potential

The electrical potential

that arises when a
dissociable salt diffuses
from a region of high
concentration to low is
the diffusion potential.

The orientation of the

diffusion potential is
such as to retard the ion
with the greater
mobility.
Membrane Potential

The diffusion potential is directly dependent on the

difference between the mobilities of the anion and cation.

R = gas constant
T = degrees Kelvin
F = Faraday constant

At 37°C, RT/F x ln {Co/Ci} simplifies to 60 (mV) x log {Co/Ci)

Membrane Potential

When Cio = Cii then V = 0

When D+ = D- then V = 0

When D+ = 0
(membrane is impermeable to the cation)

𝐶𝑖𝑜
𝑉= −60logቊ 𝑖 ቋ
𝐶𝑖
Nernst
Equation

Walter Herman Nernst (1861-1941)

Membrane Potential

1M K+ 0.1 M K+

1M Ac- 0.1 M Ac-

If the barrier (membrane) is only permeable to K+, the

electrical potential difference will be

V = 60 log {1/.1} = 60 mV

With the more dilute side (i) electrically positive

compared to the concentrated solution (o).
Membrane Potential

0.1 M K+ 0.2 M K+

0 mV -60 mV

2 fold concentration gradient pushing K+ to the

left.

60 mV electrical gradient pulling K+ to the right.

A 60 mV potential is equal to a 10 fold
concentration gradient. K+ will move from left to
right despite a concentration gradient in the
opposite direction.
Membrane Potential
Membrane Potential

Ei = (60/z) log (Cio/Cii)

Ei is the Nernst equilibrium potential for solute i in

compartment i (compartment o, the extracellular
compartment, is by convention the ground state
having a potential of 0 mV). Z = valence

Examp Co Ci Z Vm Ei C0/Ci
le
A 10 mM 100 +1 -60 mV -60 mV -1
mM -1.3
-0.69
B 5 mM 100 +1 -60 mV -78 mV
mM
C 20 mM 100 +1 -60 mV -42 mV
 Membrane Potential

Ei = (60/z) log (Cio/Cii)

EK = (60/1)log(4/139) = -92 mV

ENa = (60/1)log(145/12) = +65 mV

ECl = (60/-1)log(116/4) = -88 mV

Em = -70 mV
Range -59 to -90mV
Membrane 3.5nm
(0.07V x 3.5x10-7cm=200,000V/cm)
Transmission lines carry similar voltage/km
Membrane Potential

How membrane potential is generated and

maintained in animal cell plasma membrane?

What technique we use for measurement of ion

movement?
Membrane Potential

• The animal cell plasma membrane resting

potential is generated by the ATP-powered
Na+/K+ pump and nongated K+ channels.

• The structure and chemical nature of a channel

pore lowers the activation energy for passage of
a specific ion over other ions, which may be even
smaller.

• Patch-clamping techniques measure ion

movements through single channels.
Ion Channels: pores in membrane
Two Investigators of Pores in Cell Membranes (Peter
Agre and Roderick MacKinnon) Win Nobel Prize in
Chemistry—2003
Figure 1. Aquaporin-1, in
ribbon representation, looking
down the central channel. The
eight alpha helices that make
up each of the tetramer’s four
subunits are designated H1–
H8.
(Courtesy of Yoshinori
Fujiyoshi.)

Agr Figure 2. The narrow entrance of

e a K⊕ channel has four sites where
negatively charged oxygen atoms
stabilize K⊕ ions (green). The ions’
mutual electrostatic repulsion
pushes them through the entrance
and down into the water-filled
cavity,where water molecules
provide electrostatic
stability. (Courtesy of Roderick
MacKinnon MacKinnon.)
K+ Ion Channels

• A molecular “selectivity filter” makes ion channels selective for certain ions.
• K+ channels: tetramers of four identical subunits
• (a) Each subunit contains two conserved membrane-spanning α helices, S5 and S6, and a shorter P pore segment.
• (b) complete tetrameric channel –
• P segments –
• Connect the S5 and S6 α helices (conserved sequences in all K + channels)
• Located near the exoplasmic surface line upper part of the pore selectivity filter –
• The short α helix and an extended loop that protrudes into the narrowest part of the pore allows K + but not other ions
to pass.
• Central cavity/vestibule – lined by S6 α helices.
• Gated K+ channel (not shown):
• Opens and closes in response to specific stimuli
• Subunits contain additional gate domains
 Mechanism of ion selectivity and transport in resting K+
channels

Revise 4 Exam

Exclusion of smaller Na+ (and H+) ions:

• Selectivity filter in K+ channels – established mainly by backbone carbonyl oxygens on residues located in
a P segment Gly-Tyr-Gly sequence conserved in every known K+ channel
• (a) K+ and Na+ ions:
• Hydrated in solution
• Dehydrated in a K+ channel pore
• K+ ions passing through the selectivity filter bind to eight backbone carbonyl oxygens and lose bound
water molecules.
• Smaller Na+ ions bind poorly to the channel oxygen atoms – pass through the channel only rarely (1 in
1000)
• (b) K+ ions passing through the selectivity filter: Each non hydrated K + ion interacts with eight carbonyl oxygen
atoms lining the channel, two from each of the four subunits, replacing eight waters of hydration.
 Mechanism of ion selectivity and transport in resting K+
channels

• (c) K+ ions moving through the channel (Ion positions are numbered top to bottom from the
exoplasmic side of the channel inward.):
• State 1:
• Hydrated K+ ion – eight bound water molecules
• K+ ions at positions 1 and 3 within the selectivity filter
• Fully hydrated K+ ion within the vestibule
• State 2:
• K+ ion on the exoplasmic side of the channel has lost four of its eight waters as it enters
the channel.
• K+ ion at position 1 in state 1 has moved to position 2.
• K+ ion at position 3 in state 1 has moved to position 4.
• K+ at position 4 moves into the vestibule and picks up eight water molecules.
Patch clamping

Patch clamping records ion movements through

single ion channel.

Most cells have only one or few ion channels per

square micrometer of plasma membrane.
Ion Channel characterization

The oocyte expression assay is useful in comparing the function of normal and mutant forms of a
channel protein.
• Frog oocyte: experimental model system
• No endogenous channels
• Express encoded channels when microinjected with mRNA
• Large size makes patch-clamping studies technically easier to perform than on smaller cells.
ATP-powered pump

Four classes of ATP-powered pump:

• P-class pump
• V-class pump
• F-class pump
• ABC superfamily of proteins
Summary:
What we learned :
 Membrane transport
Three main types
Passive diffusion
Facilitated
diffusion
Active transport
Three main classes of membrane protein transport

Exterior

Cytosol

ATP – Ion
powered channels Transporters
Pumps
107 – 108 102 – 104
100 – 103 ions/sec molecules/
ions/sec sec
Membrane transport
Channels and transporter
Uniporter are faster and specific.

Low Km of GLUT1 (1.5mM) enables to transport glucose into most

mammalian cells.

Ion channels permit movement of ions down their gradient

without using energy. Channels are gated or non gated
(reversible).

ATP powered pump expend energy to move ions against their

gradient. Energy comes from ATP hydrolysis.

Uniporter transport a specific molecule down its concentration

gradient.

Cotransporters (symporter and antiporter) move specific

molecules against their concentration gradient by using the
energy supplied by one or more ions moving down their pre-
existing electrochemical gradient.
Channels and transporter
• Membrane potential is generated due to movement of ions.
• Electrical potential is measured by Nernst equation.
• The animal cell plasma membrane resting potential is
generated by the ATP-powered Na+/K+ pump and nongated K+
channels.
• The structure and chemical nature of a channel pore lowers the
activation energy for passage of a specific ion over other ions,
which may be even smaller.
• Patch-clamping techniques measure ion movements through
single channels
• A molecular “selectivity filter” makes ion channels selective for
certain ions.
• Selectivity filter in K+ channels is established mainly by
backbone carbonyl oxygens on residues located in a P segment
Gly-Tyr-Gly sequence conserved in every known K+ channel.
• Oocyte expression assay is useful tool for comparing the